Dissecting PKA activation of mTORC1 and its function in adipose tissue

剖析 mTORC1 的 PKA 激活及其在脂肪组织中的功能

• 批准号: 10091138
• 负责人: SHEILA COLLINS
• 金额: $ 6.52万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-22 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10091138
• 关键词: Adipocytes; Adipose tissue; Alanine; Amino Acids; Antibodies; Area; Aspartate; Behavioral; Biochemical; Biogenesis; Biological; Body fat; Body mass index; Brown Fat; CRISPR/Cas technology; Calories; Catecholamines; Cell Culture Techniques; Cell model; Cells; Chronic Disease; Clinical; Clothing; Complex; Country; Cyclic AMP; Cyclic AMP Receptor Protein; Cyclic AMP-Dependent Protein Kinases; Data; Data Set; Desire for food; Development; Disease; Endocrine System Diseases; Energy Metabolism; Engineering; Epidemic; Event; FDA approved; FRAP1 gene; Fatty Acids; Fatty acid glycerol esters; Follow-Up Studies; Generations; Genes; Genetic; Genetic Transcription; Glucose; Goals; Growth Factor; Human; In Vitro; Insulin; Insulin Resistance; Intervention; Investigation; Knock-in; Knowledge; Laboratory Animals; Life Style; Lipolysis; Longevity; Mechanics; Metabolic; Metabolic Diseases; Metabolic syndrome; Metabolism; Mitochondria; Mitogen-Activated Protein Kinases; Modeling; Molecular; Mus; Mutation; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Overweight; PPAR gamma; Pathway interactions; Peripheral; Phosphoproteins; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Population; Process; Production; Proteins; Proteomics; Protons; Public Health; Raptors; Receptor Activation; Regulation; Resistance; Respiration; Ribosomal Protein S6 Kinase; Risk; Rodent; Role; Route; Safety; Scanning; Signal Pathway; Signal Transduction; Sirolimus; Site; Source; Stable Isotope Labeling; Sympathetic Nervous System; Techniques; Technology; Testing; Therapeutic; Thermogenesis; Tissues; Weight Gain; Work; absorption; beta-adrenergic receptor; cell type; comorbidity; comparative; cost; experimental study; fat burning; fighting; gain of function; gain of function mutation; health economics; improved; in vivo; inhibitor/antagonist; insight; insulin sensitivity; kinase inhibitor; loss of function mutation; novel; novel strategies; obesity treatment; p38 Mitogen Activated Protein Kinase; phosphoproteomics; prevent; programs; reconstitution; transdifferentiation; uncoupling protein 1

Project Summary: Obesity is at epidemic proportions in the US. Over 60% of the population is either overweight (Body Mass Index [BMI] ≥25 to <30 kg/m2) or obese (BMI ≥30 kg/m2), placing them at risk for a large number of chronic diseases, including insulin resistance, metabolic syndrome, and type 2 diabetes. The annual costs of obesity exceed $100 billion, making it one of the most significant public health and economic issues facing the country. Unfortunately, the treatment of obesity is unsatisfactory. Lifestyle and behavioral approaches have a modest, and often transient, effect while FDA-approved therapeutic options targeting appetite or fat absorption have poor tolerability and, in some cases, safety concerns. Thus, there is a critical need for novel approaches to treat obesity. Agents acting via peripheral mechanisms to increase energy expenditure would be valuable. The sympathetic nervous system (SNS) is well-known as an activator of brown adipose tissue (BAT) and the “browning” of cells in white adipose tissue (WAT) depots to increase uncoupled mitochondrial respiration and energy expenditure. Our earlier work established a signaling cascade from β-adrenergic receptors (βARs)  cAMP  protein kinase A (PKA)  p38 MAP kinase (MAPK) to drive the transcription of brown adipocyte genes such as uncoupling protein-1 (UCP1), PPAR-gamma coativator-1α (PGC-1α), and the broader program of mitochondrial biogenesis. We have discovered that the mTOR complex-1 (mTORC1) components mTOR and Raptor are phosphorylated by PKA. This is a highly novel observation, since the `canonical' pathway to mTORC1 is through growth factors and insulin. From in vivo studies in mice we find that blockade of mTORC1 with rapamycin, or genetic deletion of Raptor specifically in adipose tissue, suppresses the ability of the βAR pathway in increase the amount of UCP1-expressing `beige' adipocytes within white fat depots, and dampens UCP1 expression and respiration in interscapular brown fat. The ability of laboratory animals and humans to expand these `beige' adipocytes is closely correlated with resistance to weight gain and improved insulin sensitivity. We have identified the phosphorylation sites on mTOR and Raptor and propose to test the physiological consequences of cells and mice in which Ser791 of Raptor is changed to either Alanine (loss-of-function mutation) or Aspartate (gain-of-function) by engineering a `knock-in' genetic construct. Since we now show that there are two distinct routes to activation of mTORC1, we are also taking an unbiased proteomic approach to identify the unique phosphorylation substrates of mTOR resulting from PKA activation vs insulin. Altogether, these experiments will shed important mechanistic and physiological insight into the steps needed for `beige' cell expansion, as well as the broader ability of the PKA mTORC1 pathway to function in other cell types.

项目概要： 肥胖在美国已成为流行病。超过60%的人口超重（体重 指数[BMI] ≥25至<30 kg/m2）或肥胖（BMI ≥30 kg/m2），使他们处于大量慢性 这些疾病包括胰岛素抵抗、代谢综合征和2型糖尿病。肥胖的年度成本 超过1000亿美元，使其成为该国面临的最重要的公共卫生和经济问题之一。 不幸的是，肥胖症的治疗并不令人满意。生活方式和行为方法有一个适度的， 而且通常是短暂的，而FDA批准的针对食欲或脂肪吸收的治疗选择， 耐受性差，在某些情况下，还有安全性问题。因此，迫切需要新的方法来 治疗肥胖症。通过外周机制起作用以增加能量消耗的试剂将是有价值的。的 众所周知，交感神经系统（SNS）是棕色脂肪组织（BAT）的激活剂， 白色脂肪组织（WAT）贮库中细胞的“布朗宁”，以增加非偶联线粒体呼吸， 能量消耗我们早期的工作建立了β-肾上腺素能受体（β AR）的信号级联反应， cAMP介导的蛋白激酶A（PKA）介导的p38 MAP激酶（MAPK）驱动棕色脂肪细胞的转录 解偶联蛋白-1（UCP 1）、PPAR-gamma coativator-1α（PGC-1α）等基因，以及更广泛的项目 线粒体的生物发生。 我们已经发现mTOR复合物-1（mTORC 1）组分mTOR和Raptor被磷酸化， PKA的。这是一个非常新颖的观察，因为mTORC 1的“经典”途径是通过生长 因子和胰岛素。从小鼠体内研究中，我们发现用雷帕霉素阻断mTORC 1，或遗传阻断mTORC 1， 脂肪组织中Raptor的特异性缺失，抑制了βAR途径增加βAR表达的能力。 在白色脂肪库中表达UCP 1的“米色”脂肪细胞的量，并抑制UCP 1表达， 肩胛间棕色脂肪呼吸。实验室动物和人类扩大这些“米色”的能力 脂肪细胞与抵抗体重增加和改善胰岛素敏感性密切相关。 我们已经确定了mTOR和Raptor上的磷酸化位点，并建议测试生理学上的差异。 Raptor的Ser 791被改变为丙氨酸（功能丧失）的细胞和小鼠的结果 突变）或天冬氨酸（功能获得）。既然我们现在表明， mTORC 1的激活有两种不同的途径，我们也采取了一种无偏见的蛋白质组学方法， 鉴定由PKA活化相对于胰岛素引起的mTOR的独特磷酸化底物。总的来说， 这些实验将揭示重要的机制和生理洞察力的步骤所需的“米色” 细胞扩增，以及PKA mTORC 1通路在其他细胞类型中发挥作用的更广泛能力。